A new stage of development begins with the formation of synaptic contacts between growing axons and their synaptic partners. Once these synaptic contacts are made, the neurons are partly dependent on the presence of their partners for continued survival and differentiation. In the absence of the synaptic targets the axons and dendrites of developing neurons atrophy and the nerve cells may die. The long-term dependency between neurons and their target is called trophic interaction. Neurotrophic factors are signalling molecules provided to neurons by trophic interaction. Neurotrophic factors originate from target tissues and regulate neuronal survival and subsequent growth and differentiation. The two major functions of neurotrophic signalling are the survival of a subset of neurons from a considerably larger population, and the formation of appropriate numbers of connections. The current neurotrophic hypothesis makes a number of assumptions about neurons and their targets. First, neurons depend on the availability of a minimum concentration of trophic factor for survival and subsequently for the persistence of their target connections. Secondly, target tissues synthesise and make available to developing neurons appropriate trophic factors. Third, targets produce trophic factors in limited amounts. Consequently the survival of developing neurons (and later, the persistence of neuronal connections) depends upon neuronal competition for available neurotrophic factor. It is now known that neurotrophins are a family of related trophic proteins. This family consists of four members in mammals that share high homology to each other (about 50% amino acid identity).
The first identified neurotrophic factor, nerve growth factor (NGF) mediates cell survival among two specific neuronal populations in birds and mammals (sympathetic and a sub-population of sensory ganglion cells). Research has shown the death of the relevant neurons in the absence of NGF, survival of a surplus of neurons in the presence of elevated levels of factor; the presence and production of NGF in neuronal targets; and the existence of receptors for NGF in innervating nerve terminals. These observations now define the criteria that must be satisfied to conclude that a factor is a neurotrophin.
Other known members of the neurotrophin family include brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and NT-4/5. Another neurotrophin, NT-6 has been discovered from the platfish Xiphophorus maculatus and Xiphoporus Lellen (Gotz et al, 1994).
Neurotrophins bind to two classes of receptors: the p75 receptor binds to neurotrophic with low affinity whilst higher affinity binding is observed with a family of receptor tyrosine kinases called Trks. This family of receptors currently contains three proteins designated TrkA, TrkB and TrkC.
The expression of a particular Trk receptor confers on the neuron the capacity to respond to the corresponding neurotrophin. Also, since neurotrophins and Trk receptors are expressed only in certain cell types in the nervous system, the binding between the neurotrophic factor and receptor accounts for the specificity of neurotrophic interactions.
Binding of neurotrophic factors to their specific receptors result in the phosphorylation of the Trk receptor.
NGF specifically activates TrkA, BDNF and NT-4/5 interact with TrkB whilst NT-3 preferentially activates TrkC.
All the neurotrophins are basic proteins of about 120 amino acids being processed from larger precursors. There are some structural features common to all members of the neurotrophin family. These include encoding of the whole precursor protein in a single exon, the basic amino acids at the end of the pre-pro region that are important in proteolytic cleavage during processing of the precursor molecule, and the presence of six cysteine residues that are thought to maintain three-dimensional conformation of the molecule by disulfide bond formation. Moreover, there are conserved regions, mainly around the cysteine residues, that contain similar amino acid sequences in all neurotrophins. Taken together, these structural features define useful criteria in the identification of novel neurotrophins.
Alignment of amino acid sequence with other neurotrophins suggests that NT6 is structurally more related to NGF. However, NT-6 contains an additional feature not shared by all other known neurotrophins: an insertion of 22 amino acids between the second and third cysteine residues of the mature molecule which contains the heparin-binding domain. Although NT-6 is able to support the survival of chick sympathetic and dorsal root ganglion neurons, its potency is much lower than that of mouse NGF, probably reflecting low conservation of the molecule during evolution.
It is an object of the invention to provide a novel neurotrophin.